Novel technology for determining the nucleotide sequence of DNA or RNA has been developed.
Today, with generally used methods that employ electrophoresis, a cDNA fragment sample synthesized in advance through reverse transcription reaction of a DNA fragment or an RNA sample for sequencing is prepared, electrophoresis is performed after conducting a dideoxy reaction by the well-known Sanger method, and the molecular weight separation/development pattern is determined and analyzed.
Meanwhile, in recent years, a method for determining the sequence information of many fragments in parallel by immobilizing many DNA fragments as samples on a substrate has been suggested. (Patent Documents 1 to 3 and Non-patent Documents 1 to 8).
Non-Patent Document 1 describes PCR performed on microparticles used as carriers carrying DNA fragments. After PCR, microparticles carrying PCR-amplified DNA fragments are introduced into many wells provided on a plate, which have diameters adjusted to the sizes of the microparticles, followed by pyrosequencing.
In addition, Non-Patent Document 2 describes PCR performed on microparticles used as carriers carrying DNA fragments. After PCR, microparticles are spread and immobilized on a glass substrate. An enzymatic reaction (ligation) is conducted on the glass substrate. Sequence information about each fragment is obtained by incorporating labeled nucleotides into the fragment and detecting fluorescence.
Further, Non-Patent Document 3 describes that many DNA probes having identical sequences are immobilized in advance on a substrate. In addition, after a DNA sample is sheared, a DNA probe sequence and an adapter sequence complementary to the DNA probe sequence are added to one end of each DNA fragment. These are hybridized on a substrate and thus sample DNA fragments are individually (one molecule by one molecule) immobilized at random on the substrate. In such case, a DNA extension reaction is conducted on the substrate to incorporate in advance labeled nucleotides, followed by washing of unreacted nucleotides and fluorescence detection. Thus, sequence information about each sample DNA is obtained.
As described above, methods for determining the sequence information of many fragments in parallel by immobilizing many nucleic acid fragment samples on a substrate have been developed. Such methods are being put into practical use.
Patent Document 1 discloses a method for immobilizing nucleic acid samples to a reaction device for nucleic acid analysis, which is used for a method for determining a lot of sequence information in parallel. The reaction device for nucleic acid analysis comprises a substrate having a sample-immobilizing layer and a spacer without a central part (the central part having been hollowed out) installed in a chamber, wherein the upper part of the chamber, the spacer, and the substrate are pressure-bonded by pressurization. A hollow part formed by the flat plate of the upper part of the chamber and the spacer and the flat plate part of the substrate form a flow channel. Injection and discharge of a solution is carried out via two tubes at the upper part of the chamber. The flat plate of the upper part of the chamber and the flat plate part of the substrate are always pressurized during use, thereby preventing leakage from the interface between the flat plate of the upper part of the chamber and the spacer, and the interface between the spacer and the flat plate part of the substrate.
The sample-immobilizing layer of Patent Document 1 improves the adhesive property between a nucleic acid sample and a substrate. A functional group is introduced into a nucleic acid sample, the functional group introduced into the nucleic acid sample interacts with a functional group of the sample-immobilizing layer, so as to improve the adhesive property. As a sample-immobilizing layer, organic material such as acrylamide, polylysine, streptavidin, or a silanized layer is used. A method using a silanized layer involves introducing a functional group such as an amino group, a carboxylic acid, or an aldehyde group into an immobilizing layer. Through the use of an arbitrary crosslinking reagent, a functional group introduced into a sample-immobilizing layer reacts with a functional group to be introduced into a nucleic acid sample, so as to immobilize the nucleic acid sample. As a functional group to be introduced into a nucleic acid sample, acrydite, biotin, amino groups, and the like are disclosed.